Abstract: ［Abstract］ Objective Wnt pathway activation exists in most colorectal cancer. Basic research has found that Wnt pathway can affect tumor immunity， but its role in colon cancer is unknown. This study focused on the effects of Wnt pathway activation on the immune microenvironment of colon cancer in order to find new targets for immunotherapy of colorectal cancer. Methods Immunofluorescence was used to verify the Wnt pathway status of SW480 and NCM460.The expressions of IL?1β in Wnt?activated colon cancer cell line SW480 and normal intestinal epithelial cell line NCM460 were compared by qPCR and Western blot. Human peripheral blood mononuclear cells were isolated and induced to form immature dendritic cells （iDC） by colony stimulating factor （GM?CSF） and IL?4， and then stimulated to induce mature dendritic cells （mDC） by using IL?1β. Expression of mature molecules on the surface of mDC cells was detected by flow cytometry. Results SW480 cells expressed beta?catenin in the nucleus， suggesting activation of the Wnt pathway. IL?1β mRNA and protein expressions increased after treatment with SW480 using Wnt pathway inhibitor （ICG?001） and increased with increasing concentration， IL?1β mRNA and protein expressions were lower in the NCM460 treated with wnt pathway activator （Licl）， and decreased with increasing concentration， and the expressions of IL?1β protein were negatively correlated with the expressions of non?phosphorylated β?catenin protein. IL?1β can promote iDC maturation， and the expression rates of HLA?DR and CD86 in IL?1β 50 ng/ml group were （69±3.62）% and （84.3±4.7）%， respectively， and the expression was significantly up?regulated compared to the RPMI group and the TNF?a 10 ng/ml group （P<0.001）. Conclusion Wnt pathway activation in colorectal cancer may inhibit the maturation of DC in tumor microenvironmentby reducing the expression of IL?1β.

Key words: wnt pathway, colorectal cancer, dendritic cells, immunity therapy, IL?1β

摘要： ［摘要］ 目的 大部分肠癌存在Wnt通路激活，基础研究发现Wnt通路可影响肿瘤免疫，但其在结肠癌中的作用不明。本研究主要探讨Wnt通路激活对结肠癌免疫微环境的影响以期发现结直肠癌免疫治疗新靶点。方法 免疫荧光技术验证SW480和NCM460 Wnt通路状态；qPCR和Western blot技术对比Wnt通路激活的结肠癌细胞系SW480和正常肠上皮细胞NCM460 IL?1β表达水平的不同；分离人外周血单核细胞，用集落刺激因子（GM?CSF）和IL?4联合诱导形成未成熟树突细胞（iDC），再加入IL?1β刺激诱导成熟树突细胞（mDC），用流式细胞技术检测mDC细胞表面成熟分子表达量。结果 SW480细胞核表达beta?catenin，提示Wnt通路激活。使用Wnt通路抑制剂（ICG?001）处理SW480后IL?1β mRNA和蛋白表达水平均升高，且随浓度升高而升高，使用wnt通路激活剂（Licl）处理NCM460后IL?1βmRNA和蛋白表达水平均低于对照组，且随浓度升高而降低，且IL?1β蛋白表达量均与非磷酸化β?连环蛋白（active β?catenin）表达量呈负相关关系。IL?1β可促进iDC成熟，IL?1β 50 ng/mL组HLA?DR、CD86表达率分别为（69±3.62）%、（84.3±4.7）%，与RPMI组及TNF?a 10 ng/mL组相比，表达显著上调（P<0.001）。结论 Wnt通路激活的结直肠癌可能通过减少IL?1β的表达来阻碍肿瘤微环境中DC的成熟。

关键词: 树突细胞, 免疫治疗, 结直肠癌, Wnt通路, IL?1β